The effector-triggered immunity landscape of tomato against Pseudomonas syringae.

Tomato (Solanum lycopersicum) is one of the world's most important food crops, and as such, its production needs to be protected from infectious diseases that can significantly reduce yield and quality. Here, we survey the effector-triggered immunity (ETI) landscape of tomato against the bacterial pathogen Pseudomonas syringae. We perform comprehensive ETI screens in five cultivated tomato varieties and two wild relatives, as well as an immunodiversity screen on a collection of 149 tomato varieties that includes both wild and cultivated varieties. The screens reveal a tomato ETI landscape that is more limited than what was previously found in the model plant Arabidopsis thaliana. We also demonstrate that ETI eliciting effectors can protect tomato against P. syringae infection when the effector is delivered by a non-virulent strain either prior to or simultaneously with a virulent strain. Overall, our findings provide a snapshot of the ETI landscape of tomatoes and demonstrate that ETI can be used as a biocontrol treatment to protect crop plants.

Predictive modeling of Pseudomonas syringae virulence on bean using gradient boosted decision trees.

Pseudomonas syringae is a genetically diverse bacterial species complex responsible for numerous agronomically important crop diseases. Individual P. syringae isolates are assigned pathovar designations based on their host of isolation and the associated disease symptoms, and these pathovar designations are often assumed to reflect host specificity although this assumption has rarely been rigorously tested. Here we developed a rapid seed infection assay to measure the virulence of 121 diverse P. syringae isolates on common bean (Phaseolus vulgaris). This collection includes P. syringae phylogroup 2 (PG2) bean isolates (pathovar syringae) that cause bacterial spot disease and P. syringae phylogroup 3 (PG3) bean isolates (pathovar phaseolicola) that cause the more serious halo blight disease. We found that bean isolates in general were significantly more virulent on bean than non-bean isolates and observed no significant virulence difference between the PG2 and PG3 bean isolates. However, when we compared virulence within PGs we found that PG3 bean isolates were significantly more virulent than PG3 non-bean isolates, while there was no significant difference in virulence between PG2 bean and non-bean isolates. These results indicate that PG3 strains have a higher level of host specificity than PG2 strains. We then used gradient boosting machine learning to predict each strain's virulence on bean based on whole genome k-mers, type III secreted effector k-mers, and the presence/absence of type III effectors and phytotoxins. Our model performed best using whole genome data and was able to predict virulence with high accuracy (mean absolute error = 0.05). Finally, we functionally validated the model by predicting virulence for 16 strains and found that 15 (94%) had virulence levels within the bounds of estimated predictions. This study strengthens the hypothesis that P. syringae PG2 strains have evolved a different lifestyle than other P. syringae strains as reflected in their lower level of host specificity. It also acts as a proof-of-principle to demonstrate the power of machine learning for predicting host specific adaptation.

Diversity, Evolution, and Function of Pseudomonas syringae Effectoromes.

Pseudomonas syringae is an evolutionarily diverse bacterial species complex and a preeminent model for the study of plant-pathogen interactions due in part to its remarkably broad host range. A critical feature of P. syringae virulence is the employment of suites of type III secreted effector (T3SE) proteins, which vary widely in composition and function. These effectors act on a variety of plant intracellular targets to promote pathogenesis but can also be avirulence factors when detected by host immune complexes. In this review, we survey the phylogenetic diversity (PD) of the P. syringae effectorome, comprising 70 distinct T3SE families identified to date, and highlight how avoidance of host immune detection has shaped effectorome diversity through functional redundancy, diversification, and horizontal transfer. We present emerging avenues for research and novel insights that can be gained via future investigations of plant-pathogen interactions through the fusion of large-scale interaction screens and phylogenomic approaches.

RecPD: A Recombination-aware measure of phylogenetic diversity.

A critical step in studying biological features (e.g., genetic variants, gene families, metabolic capabilities, or taxa) is assessing their diversity and distribution among a sample of individuals. Accurate assessments of these patterns are essential for linking features to traits or outcomes of interest and understanding their functional impact. Consequently, it is of crucial importance that the measures employed for quantifying feature diversity can perform robustly under any evolutionary scenario. However, the standard measures used for quantifying and comparing the distribution of features, such as prevalence, phylogenetic diversity, and related approaches, either do not take into consideration evolutionary history, or assume strictly vertical patterns of inheritance. Consequently, these approaches cannot accurately assess diversity for features that have undergone recombination or horizontal transfer. To address this issue, we have devised RecPD, a novel recombination-aware phylogenetic-diversity statistic for measuring the distribution and diversity of features under all evolutionary scenarios. RecPD utilizes ancestral-state reconstruction to map the presence / absence of features onto ancestral nodes in a species tree, and then identifies potential recombination events in the evolutionary history of the feature. We also derive several related measures from RecPD that can be used to assess and quantify evolutionary dynamics and correlation of feature evolutionary histories. We used simulation studies to show that RecPD reliably reconstructs feature evolutionary histories under diverse recombination and loss scenarios. We then applied RecPD in two diverse real-world scenarios including a preliminary study type III effector protein families secreted by the plant pathogenic bacterium Pseudomonas syringae and growth phenotypes of the Pseudomonas genus and demonstrate that prevalence is an inadequate measure that obscures the potential impact of recombination. We believe RecPD will have broad utility for revealing and quantifying complex evolutionary processes for features at any biological level.

Comparative genomic insights into the epidemiology and virulence of plant pathogenic pseudomonads from Turkey.

Pseudomonas is a highly diverse genus that includes species that cause disease in both plants and animals. Recently, pathogenic pseudomonads from the Pseudomonas syringae and Pseudomonas fluorescens species complexes have caused significant outbreaks in several agronomically important crops in Turkey, including tomato, citrus, artichoke and melon. We characterized 169 pathogenic Pseudomonas strains associated with recent outbreaks in Turkey via multilocus sequence analysis and whole-genome sequencing, then used comparative and evolutionary genomics to characterize putative virulence mechanisms. Most of the isolates are closely related to other plant pathogens distributed among the primary phylogroups of P. syringae, although there are significant numbers of P. fluorescens isolates, which is a species better known as a rhizosphere-inhabiting plant-growth promoter. We found that all 39 citrus blast pathogens cluster in P. syringae phylogroup 2, although strains isolated from the same host do not cluster monophyletically, with lemon, mandarin orange and sweet orange isolates all being intermixed throughout the phylogroup. In contrast, 20 tomato pith pathogens are found in two independent lineages: one in the P. syringae secondary phylogroups, and the other from the P. fluorescens species complex. These divergent pith necrosis strains lack characteristic virulence factors like the canonical tripartite type III secretion system, large effector repertoires and the ability to synthesize multiple bacterial phytotoxins, suggesting they have alternative molecular mechanisms to cause disease. These findings highlight the complex nature of host specificity among plant pathogenic pseudomonads.

Evolutionary and structural analyses of SARS-CoV-2 D614G spike protein mutation now documented worldwide.

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was declared on March 11, 2020 by the World Health Organization. As of the 31st of May, 2020, there have been more than 6 million COVID-19 cases diagnosed worldwide and over 370,000 deaths, according to Johns Hopkins. Thousands of SARS-CoV-2 strains have been sequenced to date, providing a valuable opportunity to investigate the evolution of the virus on a global scale. We performed a phylogenetic analysis of over 1,225 SARS-CoV-2 genomes spanning from late December 2019 to mid-March 2020. We identified a missense mutation, D614G, in the spike protein of SARS-CoV-2, which has emerged as a predominant clade in Europe (954 of 1,449 (66%) sequences) and is spreading worldwide (1,237 of 2,795 (44%) sequences). Molecular dating analysis estimated the emergence of this clade around mid-to-late January (10-25 January) 2020. We also applied structural bioinformatics to assess the potential impact of D614G on the virulence and epidemiology of SARS-CoV-2. In silico analyses on the spike protein structure suggests that the mutation is most likely neutral to protein function as it relates to its interaction with the human ACE2 receptor. The lack of clinical metadata available prevented our investigation of association between viral clade and disease severity phenotype. Future work that can leverage clinical outcome data with both viral and human genomic diversity is needed to monitor the pandemic.

A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries.

In bacteria functionally related genes comprising metabolic pathways and protein complexes are frequently encoded in operons and are widely conserved across phylogenetically diverse species. The evolution of these operon-encoded processes is affected by diverse mechanisms such as gene duplication, loss, rearrangement, and horizontal transfer. These mechanisms can result in functional diversification, increasing the potential evolution of novel biological pathways, and enabling pre-existing pathways to adapt to the requirements of particular environments. Despite the fundamental importance that these mechanisms play in bacterial environmental adaptation, a systematic approach for studying the evolution of operon organization is lacking. Herein, we present a novel method to study the evolution of operons based on phylogenetic clustering of operon-encoded protein families and genomic-proximity network visualizations of operon architectures. We applied this approach to study the evolution of the synthase dependent exopolysaccharide (EPS) biosynthetic systems: cellulose, acetylated cellulose, poly-ß-1,6-N-acetyl-D-glucosamine (PNAG), Pel, and alginate. These polymers have important roles in biofilm formation, antibiotic tolerance, and as virulence factors in opportunistic pathogens. Our approach revealed the complex evolutionary landscape of EPS machineries, and enabled operons to be classified into evolutionarily distinct lineages. Cellulose operons show phyla-specific operon lineages resulting from gene loss, rearrangement, and the acquisition of accessory loci, and the occurrence of whole-operon duplications arising through horizonal gene transfer. Our evolution-based classification also distinguishes between PNAG production from Gram-negative and Gram-positive bacteria on the basis of structural and functional evolution of the acetylation modification domains shared by PgaB and IcaB loci, respectively. We also predict several pel-like operon lineages in Gram-positive bacteria and demonstrate in our companion paper (Whitfield et al PLoS Pathogens, in press) that Bacillus cereus produces a Pel-dependent biofilm that is regulated by cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP).

Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster.

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.

Global landscape of cell envelope protein complexes in Escherichia coli.

Bacterial cell envelope protein (CEP) complexes mediate a range of processes, including membrane assembly, antibiotic resistance and metabolic coordination. However, only limited characterization of relevant macromolecules has been reported to date. Here we present a proteomic survey of 1,347 CEPs encompassing 90% inner- and outer-membrane and periplasmic proteins of Escherichia coli. After extraction with non-denaturing detergents, we affinity-purified 785 endogenously tagged CEPs and identified stably associated polypeptides by precision mass spectrometry. The resulting high-quality physical interaction network, comprising 77% of targeted CEPs, revealed many previously uncharacterized heteromeric complexes. We found that the secretion of autotransporters requires translocation and the assembly module TamB to nucleate proper folding from periplasm to cell surface through a cooperative mechanism involving the ß-barrel assembly machinery. We also establish that an ABC transporter of unknown function, YadH, together with the Mla system preserves outer membrane lipid asymmetry. This E. coli CEP 'interactome' provides insights into the functional landscape governing CE systems essential to bacterial growth, metabolism and drug resistance.

Conditional Epistatic Interaction Maps Reveal Global Functional Rewiring of Genome Integrity Pathways in Escherichia coli.

As antibiotic resistance is increasingly becoming a public health concern, an improved understanding of the bacterial DNA damage response (DDR), which is commonly targeted by antibiotics, could be of tremendous therapeutic value. Although the genetic components of the bacterial DDR have been studied extensively in isolation, how the underlying biological pathways interact functionally remains unclear. Here, we address this by performing systematic, unbiased, quantitative synthetic genetic interaction (GI) screens and uncover widespread changes in the GI network of the entire genomic integrity apparatus of Escherichia coli under standard and DNA-damaging growth conditions. The GI patterns of untreated cultures implicated two previously uncharacterized proteins (YhbQ and YqgF) as nucleases, whereas reorganization of the GI network after DNA damage revealed DDR roles for both annotated and uncharacterized genes. Analyses of pan-bacterial conservation patterns suggest that DDR mechanisms and functional relationships are near universal, highlighting a modular and highly adaptive genomic stress response.

Comparative Genomics and Evolutionary Modularity of Prokaryotes.

The soaring number of high-quality genomic sequences has ushered in the era of post-genomic research where our understanding of organisms has dramatically shifted towards defining the function of genes within their larger biological contexts. As a result, novel high-throughput experimental technologies are being increasingly employed to uncover physical and functional associations of genes and proteins in complex biological processes. Through the construction and analysis of physical, genetic and metabolic networks generated for the model organisms, such as Escherichia coli, organizational principles of the genome have been deduced, such as modularity, which has important implications toward understanding prokaryotic evolution and adaptation to novel lifestyles.

Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.